1382 pCMV5-TPA-NPR1 2-(3c)-Fc-WPRE-oriP

Cloning of 1382-pCMV5-TPA-NPR1#2-(3c)-Fc-WPRE-oriP

Purpose: Cloning of 1382 pCMV5-TPA-NPR1_2-(3c)-Fc-WPRE-oriP

Date: 2012-9-29 ~

Notebook:0

Page:

Clone ID: 1382

Experimenters: Paul & Shanyi & Xiuli

Results

PCR

2012-9-29

10-01-2012-Xiuli

PCR again, 1382 1383 didn't work.

PCR again using 1379 and 1378 (make 1:20 dilution, take 1ul=50ng) as template, adding 2ul DMSO into 25 ul PCR reaction. PCR works but using 30 cycle.

2012/9/30 digestion:

Lane	1	2	3	4
Name	TagGFP	1369	1376	TagGFP
Digestion	Nhe1+Not1	Nhe1+Not1	Nhe1 + Kpn1	Nhe1 + Kpn1

Colony Screening

Date: 10/7/2012

1382: vector was not digested properly

10-8-201-Xiuli

Most miniprep is vector itself,  re-screening colony using PCR methods.

2, 3 in 1382 looks correct size.

2. 3 looks right.

10-8-201-Xiuli

Re-do PCR using Kinase 7901 (B07) as template (use miniprep as template before). No. 2 in PCR reaction including 2 ul DMSO works and 25 Cycle was used for PCR.

Digest half of gel purified PCR products (10/20ul) and vector 1391 with NheI and NotI,

make ligation with  half  1382(1383) digestion and half 1391 vector piece.

10-10-2012 minipre-Xiuli

Enzyme digestion: NheI+NotI , release 3.0kb band.

All colony looks positive.